Project Summary: Serious human infections caused by Pseudomonas aeruginosa and other intrinsically antibiotic-resistant pathogens are frequent occurrences. Clinicians increasingly depend on alternative antibiotics like polymyxin (Pm) to treat such infections. Binding of Pm to lipid A, a component of the bacterial cell wall, is key to its antimicrobial mechanism of action. Recently, highly Pm-resistant (PmR) strains of P. aeruginosa have been cultured from European cystic fibrosis (CF) patients who were failing treatment with inhaled Pm. These strains have modified lipid A. Data show that in the US, ~20% of CF adults with chronic P. aeruginosa airway infection were treated with inhaled Pm during 2003-2005, most of them receiving this treatment routinely. Moreover, ~5% of CF isolates identified in US clinical microbiology labs as multidrug-resistant P. aeruginosa in the past 6 years represent PmR strains. The goal of the proposed interdisciplinary project is to define resistance mechanisms in PmR CF strains of P. aeruginosa as potential targets for drug development. The specific aims of the project are to: (1) optimize the reliability of Pm susceptibility testing for PmR strains of P. aeruginosa;(2) define the dependence of CF strain Pm resistance on specific lipid A structures and PmrAB-regulated genetic loci;and (3) define additional genetic loci required for CF strain Pm resistance. These aims will be accomplished by using the tools of analytical chemistry, mass spectrometry, bacterial genetics, and DNA sequencing to define resistance phenotypes and lipid A structures of an international collection of PmR CF strains, identify novel Pm resistance genes in these strains, and determine the dependence of resistance phenotypes on known and novel Pm resistance genes. Relevance: Because of antibiotic-resistant infections such as those caused by Pseudomonas, doctors are using alternative antibiotics like polymyxin more often. Polymyxin-resistant Pseudomonas has now been found in European cystic fibrosis patients for whom polymyxin treatment was not working, and similar bacteria have been found in cystic fibrosis patients in the US. An interdisciplinary study of polymyxin resistant Pseudomonas from patients with cystic fibrosis is proposed to pinpoint the bacterial genes responsible for this resistance, so that management strategies may be devised to overcome it.